This application relates generally to collection and amplification of circulating nucleic acids (CNAs) from a biological sample. More particularly, the application relates to separation, collection, amplification and further detection of circulating nucleic acids from the biological sample.
Circulating nucleic acids are released from a variety of tissues and are accumulated in bodily fluids. A variety of intact and/or fragmented nucleic acids have been identified in CNAs, including mRNA, miRNA, mitochondrial DNA, genomic DNA, and retrotransposons. CNAs are ideally suited for early detection of diseases as well as prognostic and theranostic applications. The diagnostic potential of CNAs has been demonstrated over a wide spectrum of diseases, including tumorigenesis, inflammation, myocardial infarction, autoimmune disorders and pregnancy-associated complications.
Circulating nucleic acids may be detected using minimaly invasive methods that sample bodily fluids. However, CNAs are present in very low abundance in the bodily fluids. Hence, analysis of CNAs generally often requires collection and processing of large volumes (milliliters or liters) of bodily fluids. However, many times, only very small amounts of bodily fluid sample (microliters) may be available for analysis, especially in the fields of in vitro diagnostics, pathology, and forensics. Moreover, large-volume sample collection often leads to significant set-up costs, transportation/handling costs and sample artifacts. Additionally, since CNAs are present outside of cells in bodily fluids, this circulating pool of nucleic acids can be gradually swamped out by intra-cellular DNAs or RNAs that are released through lysis of resident cells in bodily fluids. This swamping out or contamination may be a multi-parameter function of time, temperature, type of treatment for stabilization, and separation forces used for isolation of bodily fluids. These pre-analytical variables can produce undesirable genomic contamination from the resident cells that are present in the bodily fluid. For example, in whole blood samples, DNAs or RNAs may be released into plasma or serum from blood cells during storage and processing. This may interfere with the analysis of extra-cellular, circulating nucleic acids that are present in the plasma or serum. Genomic contamination of circulating nucleic acid pools may be reduced by maintaining the blood sample at 4° C. and processing the sample within 2 hours. However, such conditions are often not feasible and/or cost-effective for many applications.
Whole-genome amplification may be used expand the natural pool of circulating nucleic acids. However, prior attempts at whole-genome amplification of CNAs using multiple displacement amplification (MDA) techniques have highlighted unique challenges that are associated with the poor quality and low quantity of CNAs in the bodily fluids. Generally, by nature, CNAs are highly fragmented due to their origin from apoptotic/necrotic cells. The nucleic acid fragmentation pattern of CNAs is not ideal for conventional whole-genome amplification and thus leads to allelic drop-out and/or sequence-biased amplification patterns. Additionally, many of the conventional whole-genome amplification techniques require nanogram quantities of input nucleic acids. Hence, CNAs must be purified from large volumes of non-cellular fraction to meet these template concentration demands. In view of the above, there is a critical need for technologies that streamline the separation, collection, stabilization and/or amplification of circulating nucleic acids from a biological sample, particularly when analyzing small sample volumes containing picogram quantities of CNAs.